Method for identifying molecular marker closely linked to brown rust resistant gene locus of sugarcanes
A gene locus and molecular marker technology, applied in the field of sugarcane brown rust resistance breeding, can solve the problem that the identification results are easily affected by pathogens and environmental factors, the research on the selection of rust resistance gene markers has not yet been carried out, and it is difficult to meet the requirements of breeders for disease resistance. Breeding requirements and other issues, to achieve the effect of high accuracy, low cost and reliable use
Inactive Publication Date: 2016-06-29
FUJIAN AGRI & FORESTRY UNIV
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Problems solved by technology
At present, the screening, identification and evaluation of sugarcane rust resistance resources in my country is still limited to the observation and selection of artificial inoculation phenotypes, and the research on the selection of rust resistance gene markers has not yet been carried out.
The traditional artificial inoculation phenotype selection method for disease resistance is time-consuming and inefficient, and the identification results are easily affected by pathogens and environmental factors, making it difficult to meet the requirements of breeders for disease-resistant breeding
Method used
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Embodiment 1
[0025] Example 1 The above-mentioned 10 different sugarcane varieties were amplified by PCR using RBS primers, and the accuracy and specificity of the method of the present invention were verified by comparing different endonucleases respectively.
[0026] The nucleic acid DNA of sugarcane was extracted, PCR amplification was carried out using RBS labeled primers, and the Eppendorf Mastercycler EPPCR Thermal Cycler Model 5333 Gene Amplifier was used. 25μL PCR reaction system composition: 10×PCRBuffer (Mg 2+ ) 2.5 μL, dNTP (2.5 mmol / L) 2 μL, primer 1 and primer 2 (10 μmol / L) 0.5 μL each, ExTaq enzyme (5 U / μL) 0.125 μL, template DNA (25ng / μL) 2 μL, add sterilized double Distill water to bring the total volume to 25 µL. PCR amplification conditions: 94°C for 5min; 34 cycles of 94°C for 30S, 56°C for 30S, and 72°C for 40S; finally, extend at 72°C for 7min and store at 4°C for later use. After the reaction, the obtained PCR products were analyzed by 1.5% agarose gel electrophores...
Embodiment 2
[0030] Example 2 Using RBS primers to verify the F1 individual plants in the hybrid offspring of Guitang 35 (resistant to brown rust) and family 5 (susceptible to brown rust)
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The invention provides a method for identifying a molecular marker closely linked to a brown rust resistant gene locus of sugarcanes. The method comprises the step of carrying out amplification by utilizing a primer 1 and a primer 2 by respectively using genomic DNA of sugarcanes to be identified and POJ2878 as templates and is characterized by carrying out digestion on PCR (polymerase chain reaction) amplification products by utilizing restriction endonuclease HaeIII, detecting digested products through agarose gel electrophoresis and comparing the digested products with digested products of the brown rust resistant sugarcane variety POJ2878 to judge the disease resistance of the sugarcanes to be identified. The method can be used for map-based cloning and molecular marker-assisted selection of brown rust resistant genes of sugarcanes and has the advantages that the resistance of the sugarcanes to be identified to brown rust is forecast by detecting the brown rust resistant gene locus, thus not only saving the production cost but also greatly improving the selection efficiency and then accelerating the brown rust resistant sugarcane breeding process.
Description
technical field [0001] The invention belongs to the technical field of sugarcane molecular biology and sugarcane resistance identification. More specifically, it relates to a molecular marker identification method closely linked with the sugarcane brown rust resistance gene locus, and also relates to the application of the molecular marker in sugarcane brown rust resistance breeding. Background technique [0002] Sugarcane is the world's most important sugar crop and an important low-carbon economic crop. Sucrose has long accounted for more than 72% of the world's total sugar production. Its photosynthetic efficiency is the first among C4 crops. It has high light efficiency, giant growth, and perennial roots for many years. Cultivation and mass fixation of CO 2 And other characteristics, under suitable conditions, the sugar yield per mu can be as high as 1 ton, and it is also the cultivated crop with the highest biomass so far. my country is the third sugar producing count...
Claims
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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2600/13C12Q2521/301C12Q2565/125
Inventor 郭晋隆卢运海许莉萍王恒波阙友雄刘迪肖乃衍陈平华
Owner FUJIAN AGRI & FORESTRY UNIV